RESUMEN
This study provides a preliminary characterization of a metallothionein (MT) gene in Septifer virgatus and highlights its potential use in biomonitoring. The full-length SvMT cDNA and the complete sequence of the SvMT gene were identified using reverse transcriptase PCR coupled with the rapid amplification of cDNA ends and the primer walking method. The SvMT cDNA encodes a protein of 72 amino acids having nine classical Cys-X-Cys motifs. Moreover, the deduced amino acids contained the conserved motif (Cys-x-Cys-x(3)-Cys-Thr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys) of MT family 2. Its molecular mass and isoelectric point were estimated to be 7.01 kDa and 7.00, respectively. BLAST-based searching indicated that SvMT shared 81.0% amino acid sequence identity with Mytilus edulis MT-20-II. The SvMT gene has three coding exons and two introns. After exposure to 1 mg/L cadmium chloride, the expression of SvMT increased 15-fold by 3 days (d), with a maximum expression of 27-fold by 5 d compared with the pre-exposure level. After exposure to 2 mg/L zinc chloride, the expression of SvMT increased 2.5-fold by 3 d and 4.7-fold by 5 d compared with the pre-exposure level. A significant increase in the expression level of SvMT mRNA was observed after the exposure of S. virgatus to the combination of 0.003 mg/L cadmium chloride and 0.2 mg/L zinc chloride compared with the pre-exposure level. Our work indicates that the SvMT gene is associated with stress responses and could be a potential biomarker for marine pollution.
Asunto(s)
Metalotioneína/genética , Mytilidae/genética , Secuencia de Aminoácidos , Animales , Cloruro de Cadmio/toxicidad , Cloruros/toxicidad , ADN Complementario , Biomarcadores Ambientales , Metalotioneína/química , Metalotioneína/metabolismo , Mytilidae/efectos de los fármacos , Mytilidae/metabolismo , Contaminación Química del Agua , Compuestos de Zinc/toxicidadRESUMEN
Pituitary adenylate-cyclase activating polypeptide (PACAP) has neuroprotective and axonal guidance functions, but the mechanisms behind such actions remain unclear. Previously we examined effects of PACAP (PACAP38, 1 pmol) injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with control saline (0.9% NaCl) injection. Transcriptomic and proteomic approaches using ischemic (ipsilateral) brain hemisphere revealed differentially regulated genes and proteins by PACAP38 at 6 and 24 h post-treatment. However, as the ischemic hemisphere consisted of infarct core, penumbra, and non-ischemic regions, specificity of expression and localization of these identified molecular factors remained incomplete. This led us to devise a new experimental strategy wherein, ischemic core and penumbra were carefully sampled and compared to the corresponding contralateral (healthy) core and penumbra regions at 6 and 24 h post PACAP38 or saline injections. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to examine targeted gene expressions and the collapsin response mediator protein 2 (CRMP2) protein profiles, respectively. Clear differences in expression of genes and CRMP2 protein abundance and degradation product/short isoform was observed between ischemic core and penumbra and also compared to the contralateral healthy tissues after PACAP38 or saline treatment. Results indicate the importance of region-specific analyses to further identify, localize and functionally analyse target molecular factors for clarifying the neuroprotective function of PACAP38.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Fármacos Neuroprotectores/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , ADN Complementario/genética , Evaluación Preclínica de Medicamentos , Perfilación de la Expresión Génica , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Proteínas del Tejido Nervioso/genética , Fármacos Neuroprotectores/administración & dosificación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. To date, changes in the expression and effect of endogenous PACAP have not been clarified with respect to spinal cord injury (SCI). The aim of this study was to elucidate the expression pattern and function of endogenous PACAP on the contusion model of SCI using heterozygous PACAP knockout (PACAP(+/-)) and wild-type mice. Real-time polymerase chain reaction methods revealed that the level of PACAP mRNA increased gradually for 14 days after SCI and that PAC1R mRNA levels also increased for 7 days compared with intact control mice. PACAP and PAC1R immunoreactivities colabeled with a neuronal marker in the intact spinal cord. Seven days after SCI, PAC1R immunoreactivity was additionally co-expressed with an astrocyte marker. Wild-type mice gradually recovered motor function after 14 days, but PACAP(+/-) mice showed significantly impaired recovery from 3 days compared with wild-type mice. The injury volume at day 7 in PACAP(+/-) mice, and the number of single-stranded DNA-immunopositive cells as a marker of neuronal cell death at day 3 were significantly higher than values measured in wild-type mice. These data suggest that endogenous PACAP is upregulated by SCI and has a neuroprotective effect on the damaged spinal cord.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Astrocitos/metabolismo , Muerte Celular , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/biosíntesis , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/deficiencia , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/biosíntesis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Recuperación de la Función , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood-brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2-4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood-brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, ß-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-ß-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.
Asunto(s)
Encéfalo/metabolismo , Ciclodextrinas/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacocinética , Administración Intranasal , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Bulbo Olfatorio/efectos de los fármacos , Bulbo Olfatorio/metabolismo , alfa-Ciclodextrinas/farmacología , beta-Ciclodextrinas/farmacologíaRESUMEN
Methylmercury (MeHg) is an environmental pollutant known to cause neurobehavioral defects and is especially toxic to the developing brain. With recent studies showing that fetal exposure to low-dose MeHg causes developmental abnormalities, it is therefore important to find ways to combat its effects as well as to clarify the mechanism(s) underlying MeHg toxicity. In the present study, the effects of MeHg on cultured neural progenitor cells (NPC) derived from mouse embryonic brain were investigated. We first confirmed the vulnerability of embryonic NPC to MeHg toxicity, NPC from the telencephalon were more sensitive to MeHg compared to those from the diencephalon. Buthionine sulfoximine (BSO) which is known to inhibit glutathione synthesis accelerated MeHg toxicity. Furthermore, antioxidants such as N-acetyl cysteine and alpha-tocopherol dramatically rescued the NPC from MeHg's toxic effects. Interestingly, a 12 hr delay in the addition of either antioxidant was still able to prevent the cells from undergoing cell death. Although it is now difficult to avoid MeHg exposure from our environment and contaminated foods, taking anti-oxidants from foods or supplements may prevent or diminish the toxicological effects of MeHg.
Asunto(s)
Antioxidantes/farmacología , Contaminantes Ambientales/toxicidad , Compuestos de Metilmercurio/toxicidad , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Butionina Sulfoximina/farmacología , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diencéfalo/efectos de los fármacos , Diencéfalo/embriología , Diencéfalo/patología , Interacciones Farmacológicas , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Ratones , Ratones Endogámicos ICR , Neuronas/metabolismo , Neuronas/patología , Células Madre/metabolismo , Células Madre/patología , Telencéfalo/efectos de los fármacos , Telencéfalo/embriología , Telencéfalo/patologíaRESUMEN
Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway.